PROJECT SUMMARY/ ABSTRACT Staphylococcus aureus is a widespread human pathogen, capable of infecting nearly every organ in the human body. S. aureus is particularly adept at colonizing the bone to cause osteomyelitis. Indeed, S. aureus is the most common cause of both acute and chronic osteomyelitis. Skeletal infections are notoriously difficult to treat, requiring long-term antibiotic therapy and surgical debridement of necrotic bone and surrounding tissue. Despite proper infection management, the recurrence rate of osteomyelitis is around 30 percent, one year post-treatment. This treatment difficulty is exacerbated by widespread antimicrobial resistance of S. aureus both in the hospital and community settings, and by significant pathogen-induced bone destruction, which limits antibiotic penetration to the infectious focus. It is therefore clear that there is an overwhelming need for improved treatment options of S. aureus musculoskeletal infections. This proposal seeks to address this gap in treatment by understanding how S. aureus regulates virulence responses and metabolic adaptations to the skeletal environment to reveal novel therapeutic targets. In order to discover the genetic programs that accommodate bacterial survival in the skeletal environment, an unbiased, genome-wide approach known as transposon sequencing (TnSeq) was used, which identified the S. aureus two-component system SrrAB as essential for sustaining invasive osteomyelitis. SrrAB is known to regulate the aerobic/anaerobic shift and modulate toxin production. These characteristics are especially important in bone, which is an intrinsically hypoxic tissue and is sensitive to pathogen-induced bone destruction. Intriguingly, preliminary data indicate that SrrAB also regulates nutrient utilization programs. The ability of SrrAB to modulate nutrient utilization may be particularly important during osteomyelitis, as resident bone-forming osteoblasts and bone-resorbing osteoclasts exhibit a specialized metabolism requiring elevated glucose uptake, thereby limiting carbon availability to S. aureus during osteomyelitis. Accordingly, this proposal will investigate the SrrAB-dependent mechanisms by which S. aureus regulates virulence responses and metabolic adaptations to the hypoxic skeletal environment. The proposed Aims will test these hypotheses to determine (1) the mechanism of SrrAB control of toxin production in response to hypoxic growth and (2) the regulation of nutrient utilization by SrrAB that supports S. aureus growth in hypoxic skeletal tissues. Completion of the proposed experiments will elucidate microbial strategies of virulence regulation and nutrient acquisition during infection. These findings will have applications to antimicrobial and anti-virulence therapy development by furthering our understanding of the regulation of basic microbial processes essential for survival during invasive infection.